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Chapter 8 (Sterilization-197-220) PDF
Chapter 8 (Sterilization-197-220) PDF
Sterilization
Most industrial fermentations are carried out as pure cultures in
which only selected strains are allowed to grow. If foreign
microorganisms exist in the medium or any parts of the equipment, the
production organisms have to compete with the contaminants for the
limited nutrients. The foreign microorganisms can produce harmful
products which can limit the growth of the production organisms.
Therefore, before starting fermentation, the medium and all
fermentation equipment have to be free from any living organisms, in
other words, they have to be completely sterilized. Furthermore, the
aseptic condition has to be maintained.
(8.2)
kd = kd exp (- Ed ) (8.4)
o RT
where Ed is activation energy, which can be obtained from the slope of
the In(k d ) versus 1/ T plot. For example, the activation energy of E. coli
is 127 kcal/ gmole and that of Bacillus stearothermophilus Fs 7954 is
68.7 (Aiba et aI., 1973).
From Eqs. (8.2) and (8.4), the design criterion for sterilization V can
be defined as (Deindoerfer and Humphrey, 1959)
v = In !!JL
n Jo
t
=f kddt = kd ft exp (- Ed
0 Jo RT
)dt (8.5)
which is also known as the Del factor, a measure of the size of the job
to be accomplished. The Del factor increases as the final number of
cells decreases. For example, the Del factor to reduce the number of
cells in a fermenter from 1010 viable organisms to one is
10 10
V = In -1- = 23 (8.6)
T -- T0 + -
Hmst
--- (8.9)
c(M + mst)
b. For batch heating with a constant rate of heat flow such as
electrical heating, the linear form is used:
T -- T0 +
qTt
- (8.10)
cM
c. For batch heating with a isothermal heat source such as
steam circulation through heating coil, the exponential
form is used:
Sterilization 201
Example 8.1
A fermenter containing 40 m 3 of medium (25°C) is going to be
sterilized by the direct injection of saturated steam. The typical
bacterial count of the medium is about 5 x 1012 m-3, which needs to be
reduced to such an extent that the chance for a contaminant surviving
the sterilization is 1 in 1,000. The steam (345 kPa, absolute pressure)
will be injected with a flow rate of 5,000 kg/hr, which will be stopped
when the medium temperature reaches 122°C. During the holding
time, the heat loss through the vessel is assumed to be negligible.
After a proper holding time, the fermenter will be cooled by passing
100 m 3 /hr of 20°C water through the cooling coil in the fermenter
until the medium reaches 30°C. The coil has a heat-transfer area of
40 m 2 and for this operation the average overall heat-transfer
coefficient (U) for cooling is 2,500 kJ /hr m 2 K. The heat-resistant
bacterial spores in the med·ium can be characterized by an Arrhenius
coefficient kd of 5.7 x 1039 hr- l and an activation energy (Ed) of
2.834 x 105 kf/kmol (Deindoerfer and Humphrey, 1959). The heat
capacity and density of the medium are 4.187 kJ/kg K and 1,000 kg/m3,
respectively. Estimate the required holding time.
Solution:
The design criterion can be calculated from Eq. (8.5) as
3
V = In ~ = In [(5 x 1012 m -3)(40m )] = 39.8
n I x 10-3
202 Fundamentals of Biochemical Engineering
78.4t
=To + - - -
1+ 0.125t
The solution of the preceding equation for t when T = 395°K and
To = 298°K by using a numerical technique or a trial and error
approach yields that the time required to reach 122°C is 1.46 hrs.
Substitution of the previolls eq.uation into Eq. (8.5),
5
V = 5.7 X 10 39 fl.46 ex [-2.834 x 10 [298 + 78.4t )-l]dt
heat JO P 8.318 1 + 0.125 t
t7.
v
cool'
-_ 5 7 x 1039 1
4 38
0
. ex [ -2.834 X 10 -- -
P 8.318 [293 + 102exp(-0.531 t)
5
] l t t -17
-
.
6
Vho1d
thold = - - = -7.4
- =0. 037 h r =2. 25 mIn
.
kd 197.6
For this example, most of the sterilization was accomplished during
the heating and cooling periods.
(8.14)
204 Fundamentals of Biochemical Engineering
Dispersion _DS dCn ....... .... -OS dCn + .sL (-OS dCn ) dx
dx dx dx dx
..---.-.
dx
Fig. 8.1 Material balances around an elementary section in a holding tube.
Sterilization 205
The deviation from ideal plug flow due to the axial mixing can be
described by the dispersion model (Levenspiel, 1972). Let's look at
the differential element with a thickness dx in a holding tube as
shown in Figure 8.1. The basic material balance for the
microorganisms suspended in the medium is
In - Out - Killed by Sterilization = Accumulation (8.18)
At steady state, the accumulation term is equal to zero. The input and
output of the microorganisms into or out of the element have both a
bulk flow and an axial diffusion condition. The number of
microorganisms entering minus those leaving by bulk flow is
~1.0
Udt
0.1 -+-_.L--..L...-.L---l.-J..~..L....____L___L_L_..L_J....J..J..J+-~-L--L--L....J.....L..1..J.j
!£(o
dx
dCn ) -
dx
d(uCl1 )
dx
- kdC = 0 (8.22)
, x LiL
x =-
L
N pe = 0
N pe is known as Peclet number. When N pe = 00, it is ideal plug flow.
dC;z
- d' + N Pe (1- C')
n =0 at x' =0 (8.24)
x
dC~ = 0
a t x' = 1
dx'
The solution of Eq. (8.23) (Wehner and Wilhelm, 1956) is
, ~exp(N;e)
Cn IX'=l = 2 (CPN pe ) 2 (CPN pe ) (8.25)
(1 + cp) exp -2- - (1- cp) exp --2-
where
Example 8.2
A continuous sterilizer with a steam injector and a flash cooler will be
em2loyed to sterilize medium continuously with the flow rate of
3
2 m /hr. The time for heating and cooling is negligible with this type
of sterilizer. The typical bacterial count of the medium is about
Sterilization 207
Solution:
a. The design criterion can be calculated from Eq. (8.5) as
Therefore,
r - V - 37.2 = 0.0983 hr
hold - k - 387.6
d
The velocity of nledium is
2 m 3 /hr
u = - - - - = 245 m/hr
JE-O.I02 2 m 2
4
The length of the sterilizer is
L = liThoId = 24.1m
b. The Reynolds number for the medium flow is
N = 0.102 (245)(1000) = 6.24 x 103
Re
4
D
From Figure 8.2 0.8 for N Re = 6.24 X 103
iidt
208 Fundamentals of Biochemical Engineering
Therefore,
o ~ 0.8il dt = 20 m 2 jh
Now, substituting all the values given and calculated in this
problem to Eq. (8.25) will result in an equation with only one
unknown, L, which can be solved by using any non-linear
equation solver:
L = 26.8
Therefore, the holding section should be 26.8 m, which is 2.7 m
longer than the result from the assumption of ideal plug flow.
T H =Tc-(Tc-TH)exp(_UArcOOl) (8.27)
2 1 cW
For cooling using a countercurrent heat sink of equal flow rate and
heat capacity,
(8.28)
.
TJlmp.
= f (N N ) =
SV Re
(CfPPJ; Vo DeVop)
18,uDc' f.1
(8.29)
Fig. 8.3 Flow pattern around cylindrical fiber, showing the path of
particles collected by inertial impaction.
210 Fundamentals of Biochemical Engineering
'1int=
1
2.002- 1nN
Rec
[(1+K)ln(l+I\)- ~i~:;n (8.34)
-
17dif -
1
2.002 _ In N
[(1
+
Z) 1 (1
n +
2) 2(2 + Z)]
- 2(1 + Z)
(8.35 )
Rec
where 2 is the diffusion parameter defined as
1
2 = [224(2002 -lnNRe ) D Br
c vD
c
]3 (8.36)
v
Cn - _O-A(1-a) - -.....
1-(1
dh
) d( ~~v~)
C ~A(1-a)- Cll~+ dh A(1-a)
11 1- a 1- a dh
__Vo __ c
= I-a 11
(Adh)17 0 L
c C
(8.45)
Sterilization 213
where L is the length of cylindrical fiber per unit volume of filter bed,
which is related to the packing density a. and the average collector
diameter Dc as
rcD 2 L
a= _ c _ (8.46)
4
Simplifying Eq. (8.45) and substituting Eq. (8.46) for L gives
In Cn =
Cna
-~(~)11c
n Dc 1- a
(8.48)
where B is the filter depth. Therefore, the collection efficiency for the
filter bed can be estimated as
11/ = 1 - -Cn
Cno
= 1- exp[ - -4B- ( -a-)
n Dc 1- a
11c] (8.49)
When fibers are packed together in a filter bed, the velocity will be
increased and the flow pattern will be changed, which increases the
collection efficiency from impaction and interception. Chen (1955)
has determined fiber interference effects experimentally and
suggests
Example 8.3
A filter bed of glass fibers (Dc = 15 /.lm, the bed depth B = 10 em, and
packing density a = 0.03) is being used to sterilize air (20°C, 1 atm)
with an undisturbed upstream velocity, vo, of 10 cm/s. The air stream
contains 5,000 bacteria per cubic meter (d p = 1 /.lm and Pp = 1 g/ em ).
a. Estimate the single fiber collection efficiency by mertial
impaction, by interception, and by diffusion.
b. Estimate the single fiber collection efficiency based on
combined mechanisms by using Eq. (8.41) and Eq. (8. 42) and
compare the results.
c. Estimate the collection efficiency (1J a ) of the filter bed.
d. Show how the superficial velocity V o affects the various single
fiber collection efficiencies.
Solution:
a. The velocity within the filter void space is from Eq. (8.43)
where all units are in the cgs system. The mean free path A and
the Cunningham correction factor can be estimated from
Eq. (8.31) and Eq. (8.30) as:
hint = 1
2.002-1nN Re ,
[0 + /() In(l + /()- /(_~!~]
2(1 + 1(-)
= 0.96 x 10-4
2
= 2. 8y a Q2 77 x 10-5 cm2 / s
Ox au
Therefore, the Peclet number is
Dcv 1.5 x 10-3 (10.3) 4
N pe = - = 5 = 5.57 x 10
D Br 2.77 x 10-
The single fiber efficiency by diffusion can be estimated
from Eq. (8.37),
8.7 NOMENCLATURE
A surface area across which heat transfer occurs during
sterilization, m 2
B filter bed depth, m
Cf Cunningham correction factor, dimensionless
Sterilization 217
8.8 PROBLEMS
8.1 A fermenter containing 10 m 3 of medium (25°C) is going to be
sterilized by passing saturated steam (500 kPa, gage pressure)
through the coil in the ferrnenter. The typical bacterial count of
the medium is about 3 x 10 12 m-3, which needs to be reduced to
such an extent that the chance for a contaminant surviving the
sterilization is 1 in 100. The fermenter will be heated until the
medium reaches 115°C. During the holding time, the heat loss
through the vessel is assumed to be negligible. After the proper
holding time, the fermenter will be cooled by passing 20 m 3 /hr
of 25°C water through the coil in the fermenter until the
medium reaches 40°C. The coils have a heat-transfer area of
40 m 2 and for this operation the average overall heat-transfer
coefficient (U) for heating and cooling are 5,500 and 2,500 kJ /hr
m 2 K, respectively. The heat resistant bacterial spores in the
medium can be characterized by an Arrhenius coefficient (k d )
of 5.7 x 1039 hr-1 and an activation energy (Ed) of 2.834 x 105 kJ/kmgl
(Demdoerfer and Humphrey, 1959). The heat capacity and
density of the medium are 4.187 kJ /kgK and 1,000 kg/m 3,
respectively. Estimate the required holding time.
Sterilization 219
8.9 REFERENCES
Aiba, S., A. E. Humphrey and N. F. Millis, Biochemical Engineering (2nd ed.),
pp. 242 - 246. Tokyo, Japan: University of Tokyo Press, 1973.
Chen, C. Y., "Filtration of Aerosols by Fibrous Media," Chem. Rev. 55 (1955):
595 - 623.
Deindoerfer, F. H. and A. E. Humphrey, "Analytical Method for Calculating
Heat Sterilization Time," AppI. Micro. 7 (1959a): 256 - 264.
Deindoerfer, F. H. and A. E. Humphrey, "Principles in the Design of
Continuous Sterilizers," Appl. Micro. 7 (1959b): 264 - 270.
Felder, R. M. and R. W. Rousseau, Elementary Principles of Chemical Processes
(2nd ed.) pp. 630 - 635. New York, NY: John Wiley & Sons, 1986.
Friedlander, S. K., "Aerosol Filtration by Fibrous Filters," in Biochemical and
Biological Engineering Science, vol 1., ed. N. Blakebrough. London,
England: Academic Press, Inc., 1967, pp. 49 - 67.
Humphrey, A. E., "Air Sterilization," Adv. Appi Micro. 2 (1960): 301- 311.
Levenspiel, 0., "Longitudinal Mixing of Fluids Flowing in Circular
Pipes,"Ind. Eng. Chem. 50 (1958): 343 - 346.
Levenspiel, 0., Chemical Reaction Engineering (2nd ed.), p. 272. New York, NY:
John Wiley & Sons, 1972.
McCabe, W. L., J. C. Smith, and P. Harriott, Unit Operations of Chemical
Engineering (4th ed.), pp. 76 - 90. New York, NY: McGraw-Hill Book Co.,
1985.
Pasceri, R. E. and S. K. Friedlander, "The Efficiency of Fibrous Aerosol
Filters," Can. J. Chem. Eng., 38 (1960): 212 - 213.
Pelczar, M. J. and R. D. Reid, Microbiology, pp. 441 - 461. New York, NY:
McGraw-Hill Book Co., 1972.
Quesnel, L. B., "Sterilization and Sterility," in Basic Biotechnology, eds. J.
Bu'lock and B. Kristiansen. London, England: Academic Press, 1987, pp.
197 - 215.
Strauss, W., Industrial Gas Cleaning (2nd ed.), pp. 182, 278 - 297. Oxford,
England: Pergamon Press Ltd., 1975.
Wehner, J. F. and R, H. Wilhelm, "Boundary Conditions of Flow
Reactor," Chern. Eng. Sci. 6 (1956): 89 - 93.